We have studied early steps in homologous recombination carried out by a human recombinase activity isolated from nuclear extracts of HeLa cells and by E. coli recA protein. These early steps involve the recognition by recombinases of DNA sequence homology residing on two DNA molecules and the subsequent pairing of these sequences resulting in joint molecule formation. We have localized pairing interactions to short,defined regions of DNA <60 bp in length and have isolated stable structures free of any recombinase protein. These joint molecules have unusual thermal stabilities indicating that they are three-stranded DNA structures. Preliminary experiments indicate that a novel triple-stranded DNA in which the third strand is hydrogen-bonded to an intact duplex DNA may be an intermediate in homologous recombination. We are also examining the effects of DNA mismatches, deletions and insertions on nonenzymatic branch migration. Branch migration involves the exchange of hydrogen bonds between two identical DNA strands and is thought to play an important role in homologous recombination. we have designed a convenient and sensitive method for monitoring branch migration through short, defined sequences in order to study the effect of sequence context on branch migration.